Targeting KRAS G12C mutations in colorectal cancer

Abstract With the advent of Kirsten rat sarcoma viral oncogene homologue G12C (KRAS G12C) inhibitors, RAS is no longer considered undruggable. For the suppression of RAS, new therapeutic approaches have been suggested. However, current clinical studies have indicated therapeutic resistance after short-lived tumour suppression. According to preclinical studies, this might be associated with acquired genetic alterations, reactivation of downstream pathways, and stimulation for upstream signalling. In this review, we aimed to summarize current approaches for combination therapy to alleviate resistance to KRAS G12C inhibitors in colorectal cancer with a focus on the mechanisms of therapeutic resistance. We also analysed the relationship between various mechanisms and therapeutic resistance.


Introduction
Mutations in RAS, one of the most common human oncogenes, are found in 27% of human cancers [1]. Three homologues of the human RAS gene family have been identified: neuroblastoma RAS viral oncogene homologue (NRAS), Harvey rat sarcoma viral oncogene homologue (HRAS), and Kirsten rat sarcoma viral oncogene homologue (KRAS). These homologues encode four distinct tumour-causing guanosine triphosphatases (GTPases): NRAS, HRAS, KRASa, and KRASb; of these, KRASa and KRASb result from alternative splicing. As a switch protein, RAS cycles between an active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound state. A small GTPase from the RAS super protein family is encoded. KRAS has a molecular weight of 21.6 kDa and contains 188 acids. It is a guanine nucleotide-binding protein with GTPase activity, which can combine with GTP, GDP, and GTP enzyme. It mainly comprises three regions: G-domain, C-terminal, and C-terminal CAAX boxes. The G-domain, which contains Switch I and Switch II rings, is a highly conserved domain responsible for GDP-GTP switching [2].
The C-terminal, including the CAAX box, differs significantly between RAS family members. Notably, KRAS protein has a high affinity with GDP and GTP [3], acting as a molecular switch that cycles between the inactive state of GDP-binding protein and active state of GTP-binding protein [4]. In the active state, RAS transmits signals from the cell membrane to the nucleus, playing a crucial role in cellular proliferation, differentiation, and survival by meditating numerous downstream pathways, including the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathways [5]. The PI3K/AKT/mTOR signal can promote the growth of tumour and increase the risk of metastasis by meditating epithelial-mesenchymal transition (EMT) [6,7]. Additionally, the activated pathway can affect other oncogenes, such as doublecortin-like kinase (DCLK1), G protein-coupled receptor 56 (GPR56) and tripartite motif-containing 59 (TRIM59), and promote the migration and invasion of colorectal cancer (CRC) cells [8,9]. RAS and its downstream effectors are believed to be attractive targets for cancer therapy because of their critical role in driving the hallmarks of cancer and the frequency of mutations detected in human cancer.
Among these genes, the most oncogenic RAS mutations in humans are found in KRAS, which represents 85% of all mutations [1]. According to the data from TCGA PanCancer Atlas Studies, non-small-cell lung cancer (NSCLC), CRC, and pancreatic cancer [particularly, pancreatic duct adenocarcinoma (PDAC)] are the three most common cancers associated with KRAS mutations, with incidence of 30%, 42%, and 80%, respectively [10][11][12]. Oncogenic mutations in KRAS mostly focus on Codons 12, 13, and 61. The levels of mutation at these sites vary depending on the isoform and malignancy type. In particular, glycine residues at Codon 12 are most frequently affected by missense mutations [1]. Moreover, origin tissues can predict KRAS missense mutations; KRAS G12D accounted for 25%-40% of all KRAS mutations in CRC and PDAC, whereas KRAS G12C accounted for >40% of all KRAS mutations in lung adenocarcinoma [10,12,13] (Figure 1). The patients with mutant-KRAS CRC, with a median overall survival of 2 years, had worse overall survival progression and poorer prognosis than those with wild-type-RAS CRC [14,15]. As the first intracellular messenger of epidermal growth factor receptor (EGFR), KRAS circulates between the inactive GDP-binding protein and active GTP-binding protein [13,16]. Meanwhile, missense mutations at Codons 12, 13, and 61 block GTP hydrolysis (i.e. block progression to the inactive stage) by limiting the interaction of GTPase-activating protein (GAP) proteins with the GTPase site of RAS [17]. Subsequently, KRAS triggers multiple proliferative signalling pathways, including the mitogen-activated protein kinase (MAPK)/ERK and PI3K pathways, to promote cell growth, division, and differentiation [18][19][20]. Thus, there is a critical need to establish an effective target therapy to sustainably inhibit the reactivity of KRAS and its downstream signalling among patients with this mutation. Unfortunately, despite 40 years of research on this topic, no treatment has proven stable enough to permanently suppress KRAS mutations. Further, no drugs have shown efficacy while remaining within acceptable cytotoxic levels. Because of its affinity with GTP and the absence of significant binding sites that could accommodate allosteric inhibitors, KRAS has long been considered 'undruggable' [21,22]. As mentioned previously, for patients with KRAS mutation, treatment with EGFR or vascular endothelial growth factor (VEGF) inhibitors even combined with chemotherapy has not shown any benefit.
KRAS G12C inhibitors have demonstrated completely conceptual improvement in patients with KRAS mutation [23]. In addition, this special targeted inhibitor has a significant influence on mutated solid cancer cells. Herein, KRAS G12C inhibitors have drastically altered the behaviour of mutant cells in CRC. Further, combined therapy with other inhibitors has shown a potential inhibitory effect in CRC. Optimizing the use of combination therapy to reduce acquired resistance is vital.

Limited efficiency of KRAS G12C inhibitors
Recent attempts to suppress KRAS inhibitors that are specific to mutant subtypes have proven successful. The foundational work for therapeutic KRAS blockade was laid by a groundbreaking discovery of binding of a KRAS G12C inhibitor to the Switch II region [24]. The potential target is significant across all patients with mutant KRAS who have limited effective treatment therapies. The first KRAS G12C inhibitor targeting the inactive GDP-bound state was ARS-853, which led to the development of novel anti-RAS therapeutics [25]. Then the same team led by Janes et al. [26] developed the first G12C inhibitor in vivo known as APS1620, which overcame the shortcomings of ARS-853 regarding plasma stability and oral availability, showing higher potency and selectivity for KRAS G12C. Moreover, among several compounds with improved biological activities, adagrasib (MRTX849) and sotorasib (AMG-510) were the first drugs to enter clinical trials [23,26,27]. The trials revealed that these two drugs can reduce the viability of mutant-KRAS cells and inhibit MAPK-pathway signalling by interacting with the Switch II region [23,27,28]. Notably, among patients with NSCLC, 19 (32.2%) showed a confirmed objective response, whereas 52 (88.1%) achieved disease control [28]. The effectiveness and tolerability of sotorasib in patients with advanced or metastatic NSCLC (NCT03600883) were evaluated in the recently completed phase II CodeBreak 100 trial [29]. This trial included patients who failed at least two previous anticancer therapies and indicated that the drug was safe and tolerable, similarly to the previously reported phase I data [28]. NSCLC cases had a median progression-free survival (mPFS) of 6.3 months, diseasecontrol rate (DCR) of 88.1%, and overall response rate (ORR) of 36%. Meanwhile, the clinical trial (NCT03785249) of adagrasib is currently ongoing. The preliminary findings have demonstrated the effectiveness and tolerated drug toxicity of adagrasib, with ORR and DCR of 45% and 96%, respectively, in NSCLC.
G12C variants only account for 3% of CRC cases, indicating that they are not as abundantly expressed as observed in cases of pancreatic cancer or NSCLC [30]. For these cases, the efficacy of the KRAS G12C inhibitor was comparable to that reported in a previous study. Among patients with a subtype of CRC, 7.1% (three patients) had a confirmed response, with an mPFS of 4 months after treatment with sotorasib [28]. The data presented at the 2020 EORTC-NCI-AACR Annual Symposium supported the efficacy of adagrasib in CRC, with an ORR of 17% and DCR of 96% [31]. However, some patients experienced disease progression immediately after an initial response [28]. In addition, the clinical activity of the drug in CRC cases was reduced compared with that in NSCLC cases. These results suggest that targeting KRAS G12C should not be the only therapeutic strategy employed in a study. Combination therapy can be used for limiting adaptive feedback, similar to the experience with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors; this is essential to maximize the durability of response in each tumour type [32,33]. Importantly, KRAS G12C mutation plays a key role in colon carcinogenesis and development compared with other tumours; however, the effect of specific inhibitors remains unclear. Further research on the molecular pathways associated with KRAS G12C mutation is warranted and it is important to determine the mechanism by which various tumours respond to inhibitors. Moreover, the mechanisms of resistance to KRAS inhibitors are complex and the choice of the combination therapy needs further investigation to guide future treatment approaches.

Mechanisms of KRAS gene alteration
Preclinical studies have indicated that the therapeutic efficacy of KRAS G12C inhibitors is reduced via various potential resistance mechanisms, including innate, acquired, and adaptive tumour responses. A lack of dependency on KRAS signalling could partially account for the intrinsic resistance noted in preclinical models, which may underlie the variations in patient responsiveness [23,34,35]. In some non-KRAS-dependent cells, even when KRAS was completely inhibited, the cells were still viable, indicating that the resistance of some KRAS G12C-mutated tumours to G12C inhibitors is due to the low dependence of cells on KRAS [36].
Variation in mutant-KRAS cells may impact the therapeutic potential of KRAS G12C inhibitors. Because these inhibitors only inhibit KRAS in the inactive GDP-bound state, Xue et al. [16] attributed treatment resistance to the variation in KRAS-mutant cells. However, cells in the GTP-bound state are not sensitive to inhibitor treatment and continue to activate downstream signalling pathways, which in turn promote cell proliferation and differentiation. This may be attributed to the heterogeneous response of KRAS G12C-mutant cells to G12C inhibition by ARS-1620. The newly formed KRAS G12C remains in its active GTP-bound, drug-insensitive state under the action of EGFR and phosphatase SHP2 signalling. Upon further investigation regarding the factors responsible for the evasion of KRAS G12C suppression, heparin-binding EGF (HBEGF) and aurora kinase (AURKA) were found to play a significant role in KRAS treatment resistance through the analysis of differential expression and genome-wide knock-down screens. HBEGF potentially enhances mutant cell resistance to KRAS G12C inhibitor mediated through the EGFR pathway. HBEGF was transiently downregulated in response to ARS-162 but was rapidly upregulated after 48 h. Furthermore, silencing of HBEGF using small interfering RNA significantly enhanced the inhibitory effect of the KRAS G12C inhibitor. Moreover, AURKA may be required for reversing drug-induced quiescence; this maintains KRAS in the active GTP-bound state, which when stabilized reacts with KRAS G12C and the downstream effector CRAF. Meanwhile, there is a difference in the operating sequence of HBEGF and AURKA; HBEGF exposure is the initial stimulus and AURKA operates later.
Although these experiments were verified in lung cancer cells, they also have implications for CRC promoting. Thus, treatment resistance can result from the non-uniform stage of KRAS G12Cmutant cells.
The activation of wild RAS could also explain the resistance to specific inhibition strategies. Ryan et al. [37] studied the adaptive response of KRAS G12C-mutant lung, colon, and pancreatic cancer cells to selective KRAS G12C inhibition using the covalent KRAS G12C inhibitors ARS-1620 and AMG 510. They revealed that KRAS G12C inhibitors downregulated the MAPK pathway in all cells, as evidenced by the decreased expression of phosphorylated MEK, ERK, and receptor tyrosine kinases (RTKs) at 4 h. Despite continuous suppression of GTP-bound KRAS, MAPK-pathway reactivation was observed in most cells by 24-48 h. Isoform-specific pull-down assays revealed that treatment with KRAS G12C inhibitor resulted in a several-fold increase in NRAS GTP and HRAS GTP levels. These results suggest that KRAS G12C-mutant cells rapidly adapt to the selective suppression of mutant KRAS caused by activating wild-type RAS; further, these indicate that this oncogenic bypass was sufficient for restoring MAPK signalling. Meanwhile, Zhao et al. [38] discovered the genetic basis of the action of first-line mutant GTPase inhibitor sotorasib in samples with KRAS mutation. The resistance to KRAS (G12C) inhibition is associated with lowallele-frequency hotspot mutations in preclinical patientderived xenograft and cell models. In the single-cell sequencing of isogenic lineage, including KRAS (G12V or G13D), NRAS (Q61K or G13R), and BRAF (G596R), they could identify either or both secondary RAS and BRAF mutations (G12C) in the same cells. Further, the novel mutations could bypass the inhibition without affecting the original inactivation subgroup. This study demonstrated a heterogenous pattern of resistance with multiple sub-clonal events during G12C-inhibitor treatment.
In addition to the generation of novel KRAS and activation of wild-type informs, multiple co-mutational alterations can cause treatment resistance. Awad et al. [39] analysed KRAS G12C-mutant cells among patients who received adagrasib monotherapy. They reported putative mechanisms causing treatment resistance at the genomic and histologic levels. Among 38 patients with KRAS G12C-mutated tumours, 17 developed mutational resistance mechanisms; of 7 (18% of the cohort) patients with multiple co-mutational mechanisms, 4 (11%) had CRC. Patients with CRC had multiple genetic alterations, including acquired KRAS mutations in G12D/R/V, G13D, and H95R, as well as amplified KRAS G12C alleles. Other notable findings were the presence of multiple carcinogenic fusions involving BRAF, NRF1, and FGFR3. The acquired bypass gene mechanism included MET amplification and loss of function mutations in NF1 and PTEN.
Regarding the adaptive mutations of KRAS, different mutant sites have various degrees of resistance to KRAS inhibition. Koga et al. [40] identified 12 different secondary KRAS mutations and reported that 124 (87%) individuals had secondary KRAS mutations. For instance, Y96D and Y96S were resistant to the individual inhibitors, i.e. a new SOS1 inhibitor (BI-3406) and trametinib; however; the combination of the two inhibitors was highly effective against both mutations. G13D, R68M, A59S, and A59T, which were extremely resistant to sotorasib, were found to be sensitive to adagrasib. Q99L, which was resistant to adagrasib, remained susceptible to sotorasib. Tanaka et al. [41] investigated acquired resistance to adagrasib using circulating free DNA (cfDNA). The identical KRAS G12C and TP53 F338fs variants that appeared in patients' tumours and cfDNA before therapy were also detected after the emergence of resistance along with the introduction of 10 unique mutations affecting the four RAS/MAPK subunits, namely KRAS, NRAS, BRAF, and MAPK1. In addition, the three KRAS mutations G13D, G12V, and Y96D were detected in the cfDNA after advancement. Structural modelling revealed that the Y96D mutation destroys crucial hydrogen bonds between KRAS and the Y96D residues of adagrasib. Y96D was resistant not only to adagrasib but also to sotorasib and ARS-1620. The presence of a Switch II pocket-type mutation in Y96D may hinder the binding of KRAS G12C (OFF) inhibitors and promote tolerance to KRAS G12C (ON) inhibitors. However, this can be inhibited by a functionally unique KRAS G12C (ON) inhibitor (RM-018). Although the experiment mainly involved patients with lung cancer, differences among secondary mutant sites may similarly contribute to treatment resistance in CRC.

MAPK-pathway reactivation
The bypass MAPK pathway plays an important role in KRAS therapy as a downstream component of the RAS cascade. Similar to BRAF and EGFR, KRAS G12C inhibitors are the upstream targets of the MAPK pathway. Regarding possible mechanisms of therapeutic resistance, feedback activation of the MAPK pathway has an impact on the efficacy of specific inhibitors [16,23,27,42]. Hence, this feedback activation may cause resistance of KRAS to specific allele inhibition.
As previously observed in BRAF-mutated CRC [43], the adaptive response to RAF-inhibitor treatment was a useful reference for the development of KRAS G12C inhibitors. BRAF V600E-mutated cells treated with RAF inhibitors can result in a rapid relief of upstream feedback of RTKs and RAS. Furthermore, the dimerization of BRAF can decrease the sensitivity to RAF inhibitors and lead to the reactivation of ERK signalling [44]. Hence, combination therapies that target multiple nodes in the pathways (especially upstream nodes) can induce a more marked response, as observed in preclinical models. To confirm this hypothesis, Amodio et al. [45] examined the effects of AMG510 in CRC cells. The levels of cell basal RTK activation in CRC cells were higher and more responsive to growth factor stimulation than those in NSCLC cells. Moreover, the rapid rebound of RTKs, especially EGFR, was responsible for CRC resistance to KRAS G12C inhibitors. Combination therapy targeting both EGFR and KRAS G12C was highly effective in CRC cells. The combination of cetuximab and sotorasib effectively inhibited the activation of the EGFR-driven MAPK pathway in CRC cells, thus sustaining downstream target inhibition, significantly improving KRAS G12C inhibition, and achieving tumour regression.
The aforementioned studies demonstrate that the reactivation of RTK signalling (especially EGFR) is an important cause of treatment resistance to KRAS G12C inhibitors in CRC. Meanwhile, in patients with EGFR wild-type CRC, KRAS mutations often lead to secondary resistance resulting in inferior outcome of anti-EGFR therapy [46]. Thus, targeted therapies against EGFR have been investigated as they provide a more effective direction in clinical practice. Cetuximab monotherapy has shown favourable clinical results in EGFR-mutated lung cancer [47,48]. Further, a combination of cetuximab and fluorouracil chemotherapy has shown good results in CRC [49]. In BRAF-mutated CRC, cetuximab along with BRAF inhibitors is already the standard second-line treatment in clinical practice [43,50]. Currently, the combination of KRAS G12C and EGFR inhibitors has shown positive results in mouse models [16,27].
In vivo and in vitro experiments have demonstrated the reactivation of upstream RTKs, which may be critical for the resistance to KRAS inhibition in CRC. Furthermore, compared with NSCLC, the pronounced dependency on the EGFR/MAPK pathway in CRC may be responsible for differences in their treatments. The possible reasons why EGFR triggers resistance to KRAS G12C inhibitors are multiple. It may be related to the intrinsic RTK dependency and sensitivity of CRC, high active RTKs levels, and reactivation of downstream effectors. In these cases, a combination of KRAS inhibition and EGFR blockade can be a viable therapeutic strategy. Nevertheless, rational and tissuespecific combination therapies are necessary for precise and effective disease control. The characteristics of other KRASmutant alleles varied extensively; thus, further investigation is warranted to determine whether other alleles can benefit from EGFR-blockade combination therapy.
SHP2 plays an important role in the RAS pathway mediated by RTK phosphorylation, providing a potential target for KRAS G12C combination therapy [51]. As a mediator, it can enhance signalling through the MAPK pathway. Thus, SHP2 inhibition can block RAS activation mediated by multiple RTKs. Furthermore, as a single target, SHP2 inhibition has shown favourable efficiency in preclinical models. SHP2 inhibition can reduce the level of KRAS-GTP by meditating the disruption of SOS1 and inhibiting the activation of the MAPK cascade [52]. However, single-agent SHP2 inhibition via SHP099 is incomplete. Meagan et al. [37] investigated the interaction between SHP2 and the RAS/MAPK pathway and revealed that the cosuppression of ARS-1620 and SHP099 can lead to complete inhibition of KRAS-GTP and RAS-GTP levels in CRC cells. This suppression of the MAPK pathway by a combination of ARS-1620 and SHP099 treatment can last much longer than SHP099 by single-agent inhibition. It was suggested that SHP2 inhibition can alleviate the induction of wild-type RAS and prevent the adaptive feedback resistance to KRAS G12C inhibition.
Similarly, other trials are being conducted to assess SHP2 inhibitors alone or in combination with other drugs. SHP2 inhibitors were found to effectively inhibit RTK-mediated-pathway reactivation in KRAS-mutated cancer [53]. Currently, Mirati et al. [54] are conducting a clinical trial to combination therapy with SHP2 inhibition. The selective SHP2 inhibitor RMC-4630 showed some early evidence of efficacy in patients with KRAS G12C-mutated NSCLC, with disease control being achieved in five of seven patients (71%). Moreover, RMC-4630 is being tested in combination with the MEK inhibitor cobimetinib, which has shown some preliminary evidence of antitumour activity in KRAS-mutated CRC, as indicated by tumour reduction in 37.5% of patients (three of eight). Meanwhile, Carmine et al. [55] demonstrated that SHP2-I/G12C-I combination can not only increase the occupancy of KRAS-GDP but also alter the immune microenvironment in KRAS-mutated PDAC. Similarly, Chen et al. [56] revealed that the novel SHP2 inhibitor TNO155 effectively blocks the feedback activation induced by KRAS G12C and substantially enhances the efficacy. The combination with TNO155 improved the efficacy of mobility shift in mutant proteins as well as decreased the rebound of p-MEK and p-ERK. In vitro experiments revealed that TNO155 exerts a synergistic effect with the KRAS G12C inhibitor, which can suppress the proliferation of mutant cells. In summary, combining SHP2 with the KRAS G12C inhibitor can partially reduce treatment resistance. Clinical trials of multiple specific inhibitors in combination with SHP2 are currently underway and further investigation is also warranted to determine the ideal dosage [57].
A combination of ERK and KRAS G12C inhibitors has been used to demonstrate the role of MAPK-pathway activation in resistance to KRAS G12C inhibition. Other studies have also shown that a combination of MAPK downstream pathway inhibitors can alleviate treatment resistance. MEK, a vital part of the MAPK pathway, has previously been studied. However, several clinical experiments have shown that MEK inhibition via monotherapy has limited treatment activity in patients with metastatic KRAS-mutated CRC [58][59][60]. One of the reasons for the weak effect of MEK inhibitors in KRAS-mutated CRC may be the activation of the collateral feedback loop [61][62][63]. Meanwhile, MEK inhibition plus PD-L1 inhibition has limited therapeutic efficiency in KRAS-mutated CRC [59]. However, a combination with other downstream inhibitions can enhance the target effect of MEK inhibition. The MEK inhibitor can synergize with the RAF inhibitor by increasing the sensitivity to RAF inhibition in KRASmutated cancer [64]. Moreover, dual inhibition of SOS1 and MEK demonstrated favourable suppression in KRAS-mutated mouse models [65]. Furthermore, dimerization of mutant KRAS and wild-type KRAS may affect the sensitivity to MEK inhibitors and reducing the level of wild-type KRAS may increase the sensitivity to MEK inhibitors in KRAS-mutant cells [66,67].
KRAS has multiple downstream pathways, but inhibiting these downstream pathways is less effective than inhibiting the upfront target. This may be attributed to the complexity of the RAS pathway, in which single inhibition of a downstream target leads to the negative feedback activation of upstream targets. This activates multiple parallel signalling pathways downstream, which in turn promote tumour-cell proliferation. Hence, the combination of upstream target inhibition would be more effective than downstream inhibition alone. This strategy can block the activation of the MAPK pathway and prevent the activation of a potential parallel pathway. Further, combined inhibition of KRAS G12C and upstream targets of the MAPK pathway is crucial for minimizing treatment resistance.

Mutation of the PI3K pathway
In addition to the combined inhibition of the MAPK pathway, the PI3K pathway plays a vital role in KRAS-mutated CRC. PI3K is a cytoplasmic molecule located downstream of KRAS and is a part of the PI3K/AKT/mTOR pathway. As a second effector pathway, PI3K is also activated by KRAS, in which there are three classes of PI3K activation. Class I PI3Ks, which are activated by GTP-bound RAS, phosphorylate phosphatidylinositol 4,5bisphosphate to generate phosphatidylinositol (3,4,5)-trisphosphate. The phosphorylation of PI3K recruits AKT to the membrane and activates mTOR, which promotes cell proliferation and differentiation [68]. In contrast to the MAPK pathway, which cannot mutate simultaneously with RAS, the PI3K pathway can co-mutate with RAS [69]. In other words, when MAPK inhibitors or KRAS inhibitors are used alone, the PI3K pathway may be activated, resulting in therapeutic resistance. Aberrant activation of the PI3K pathway strongly reduces the efficiency of MAPK suppression in KRAS-mutated CRC.
Previous studies have reported that the activation of the PI3K pathway is responsible for treatment resistance to MAPKpathway inhibitors and chemotherapy [70]. Misale et al. [42] revealed that the inhibition of PI3K and G12C consistently improved the suppression efficacy of various cell lines. This effect was attributed to the reactivation of ERK signalling mediated by PI3K through junctional proteins or was due to the combined shutdown of two major cellular signalling pathways [71,72]. Some studies have demonstrated the synergistic effects of PI3K and MEK inhibitors. However, although combination therapy is more effective, it has greater toxicity [73,74]. Further studies are needed to access the toxicity profile of multidrug combinations.
Therefore, the use of the KRAS G12C inhibitor alone is likely to lead to the activation of the PI3K pathway and the combination of PI3K and KRAS G12C inhibitors may reduce treatment resistance. However, clinical trials of PI3K and KRAS G12C in CRC have not yet been conducted. The toxicity of G12CþPI3K inhibition and its efficiency in CRC remain unclear. Further studies are needed to address these aspects using more comprehensive approaches.

Mechanism of immune resistance
Canon et al. [23] revealed that AMG-510 can synergize with immunotherapy by increasing the number of infiltrating CD8 þ T cells, thus inducing a pro-inflammatory microenvironment. Notably, AMG-510 significantly inhibited the growth of CT-26 KRAS G12C cells in immunocompetent mice; however, in mice that lacked T cells, AMG-510 only induced short-lived regression but could not treat them. This suggests that the completeness of the immune system influences KRAS G12C treatment resistance. Meanwhile, combination therapy with AMG-510 and anti-programmed cell death-ligand 1 (anti-PD-L1) showed no effect on T-cell responsiveness but had higher antitumour activity than MEK inhibitor combined with anti-PD-L1. A recent study by Ghiringhelli et al. [75] revealed that the combination of MEK inhibition and chemotherapy synergized with PD-L1 blockade. Furthermore, treatment with AMG-510 enhances T-cell priming and antigen recognition of tumour cells. Neither CT-26 KRAS G12C nor CT-26 parental tumours recurred after treatment with AMG-510 and anti-PD-L1, suggesting that these agents promoted the establishment of a durable immune system. Notably, Fedele et al. [53] also found that the KRAS G12C and SHP2 inhibitors can affect the tumour microenvironment (TME) by increasing the number of CD8 þ T cells.  Meanwhile, a novel strategy using imaging mass cytometry revealed that the KRAS G12C inhibitor can remodel the TME and promote the infiltration and activation of presenting and effector cells [76]. These results suggest that KRAS G12C inhibitors can influence the TME of mutated cells. Future studies should explore more combinations of KRAS inhibitors with immunotherapy. Accordingly, more clinical trials combining sotorasib/adagrasib with anti-PD1/PD-L1 are currently underway (NCT03600883 and NCT03785249; clinicaltrials.gov).

Optimizing the KRAS G12C inhibitor
Adaptive resistance feedback to KRAS G12C can result from various mechanisms, with independent or reciprocal interactions between the different mechanisms ( Figure 2). Combination therapy is the best approach to overcome acquired therapeutic resistance and these commonly target upstream RTKs (especially EGFR reactivation in CRC), mediators of the MAPK cascade SHP2/SOS, adaptive alterations of RAS genes, and activation of multiple downstream pathways ( Figure 3). In addition, the KRAS G12C target therapy can synergize with immunotherapy by remodelling the TME and enhancing the infiltration of T cells. Meanwhile, combination therapy can reduce the expression of PD-L1 and B7-H3, which enhances the antitumour immunity [77]. Compared with NSCLC, CRC is characterized by more complex tumour heterogeneity and diverse mutation mechanisms. Therefore, combination therapy with other targets is needed to achieve better tolerability in patients. The current clinical trials analysing KRAS G12C combination therapy are summarized in Table 1.

Conclusions
Current knowledge regarding the KRAS G12C inhibitor debunks the notion that the KRAS oncogene is 'undruggable'. Numerous targeted agents that are successfully marketed have demonstrated both efficacy and tolerability [28,29]. However, in BRAFand EGFR-mutated CRC, monotherapy will ultimately lead to the emergence of drug resistance due to adaptive resistance or mutation of the gene itself. Furthermore, the resistance mechanism is tissue-specific and, among the above reasons, the activation of the upstream EGFR pathway is the primary factor in CRC. It should be emphasized that KRAS and its upstream and downstream pathways such as MAPK and PI3K are not singularly regulated, and multiple targets and downstream pathways can be mediated by negative feedback activation. This may lead to the reactivation of downstream pathways, resulting in drug resistance. Meanwhile, the variation in KRAS G12C inhibitors in diverse populations and tumour types also indicate the existence of intrinsic resistance mechanisms [78].
The commercial availability of KRAS G12C inhibitor should also be considered. G12C mutation accounts for only 3% of all mutations in patients with CRC. Some patients can benefit from this target therapy; however, acquired resistance and realworld therapeutic effects are still challenges for G12C target therapy as other patients with KRAS mutation still need more effective target methods. Therefore, searching more specific biomarkers and broader target inhibitors and comprehensively utilizing multi-pathway inhibition (e.g. co-targeting molecules in various pathways such as SOS/SHP2) are important strategies to alleviate KRAS treatment resistance, which warrants further investigation.